進階搜尋


   電子論文尚未授權公開,紙本請查館藏目錄
(※如查詢不到或館藏狀況顯示「閉架不公開」,表示該本論文不在書庫,無法取用。)
系統識別號 U0026-1009201614235500
論文名稱(中文) 自噬反應相關之Atg5基因於癌化現象所扮演之角色
論文名稱(英文) The role of Atg5 gene in tumorigenesis
校院名稱 成功大學
系所名稱(中) 微生物及免疫學研究所
系所名稱(英) Department of Microbiology & Immunology
學年度 104
學期 2
出版年 105
研究生(中文) 王尹平
研究生(英文) Yin-Ping Wang
學號 S46034093
學位類別 碩士
語文別 英文
論文頁數 49頁
口試委員 指導教授-劉校生
口試委員-張志鵬
口試委員-徐麗君
口試委員-蘇純立
中文關鍵字 自噬反應  Atg5  細胞生長  細胞爬行  腫瘤生成  Wnt5a 
英文關鍵字 Atg5  Proliferation  Migration  Tumor formation  Wnt5a 
學科別分類
中文摘要 腫瘤發生的過程稱之為癌化,是一個動態且複雜的進程,許多研究證實,自體吞噬(autophagy)可以調控癌化過程。自噬反應是一個維持細胞恆定的機制,主要是藉由降解老舊的胞器、蛋白質或是其他細胞之內的物質來保護細胞。Atg5為自噬反應過程的必要基因,在自噬小體的形成上扮演重要的角色,而越來越多的研究指出,Atg5蛋白除了參與自噬反應外,還有一些與自噬反應無關的其他功能,包括細胞凋亡、免疫反應和有絲分裂。我們實驗室已發現,提升自噬反應可以抑制膀胱癌,並伴隨著Atg5蛋白的大量,顯示自噬反應和Atg5蛋白與腫瘤的抑制有關,但單獨Atg5蛋白在沒有自噬反應的背景下對於腫瘤的影響卻是不明確的,因此在本實驗中,我們利用自噬反應缺失的小鼠纖維母細胞(Atg7-/- MEF),在其中過量表現Atg5蛋白來釐清沒有自噬反應的狀態下,Atg5蛋白對癌化進程的影響為何。實驗結果顯示,Atg5蛋白的過量表現可促進細胞增生(proliferation)及細胞爬行(migration),此外也增強了群落形成(colony formation)的能力,在NOD/SCID小鼠皮下腫瘤實驗中也發現,與控制組相比,Atg5蛋白的表達可短暫性促進腫瘤形成,但腫瘤的體積會隨著時間遞減,並在12天內消失;除此之外,我們也發現,若將自噬現象的能力回復,細胞生長和爬行有下降的趨勢,表示此Atg5蛋白的作用的確是在自噬現象缺乏下的功能,而此結果也顯示Atg5蛋白在有無自噬反應的情況下可能扮演兩種不同角色。而在路徑的探討中,我們發現Atg5蛋白單獨的過量表現會活化Wnt5a和其下游的p-JNK,並伴隨-catenin表現下降。總結,Atg5在自噬現象缺失的狀態下,可能透過Wnt5a路徑的活化來促進細胞生長和爬行,並增強腫瘤生成的能力。
英文摘要 Tumorigenesis is a dynamic and complex process of tumor development, which can be regulated by autophagy. Autophagy is a mechanism by which organelles, proteins and other cytoplasmic components are degraded to maintain cellular homeostasis. Autophagy related 5 (Atg5) is essential for autophagy and plays an important role in autophagosome formation. Increasing evidence suggests that Atg5 also plays autophagy-independent roles in various cellular functions, including apoptosis, immunity and mitosis. We previously demonstrated that activated autophagy suppressed bladder tumor formation accompanied with increased Atg5 protein expression, indicating that autophagy and/or Atg5 plays a suppressive role in tumor formation. The role of Atg5 protein affects tumorigenesis without autophagy is unclear. Therefore, we aim to reveal the autophagy-independent role of Atg5 and its underlying mechanisms in tumorigenesis. An autophagy-deficient Atg7-/- mouse embryonic fibroblast cell line was used to overexpress Atg5 transgene and two stable clones were established to explore the single gene effect of Atg5. We found that Atg5 overexpression increased cell proliferation, colony formation and migration under autophagy deprivation conditions. However, recovery of autophagy by Atg7 transfection reversed the results, implicating that Atg5 might have dual roles in the absence and the presence of autophagy. In vivo animal experiment also showed that Atg5 improved tumor formation in subcutaneous, but the tumor failed to progress and regressed within twelve days. Moreover, we further explored the underlying mechanism and found the increased expression of secreted Wnt5a and p-JNK, but the decreased expression of-catenin. In summary, Atg5 promotes tumorigenesis in vitro and in vivo in an autophagy-independent manner.
論文目次 中文摘要 I
Abstract III
致謝 V
Contents VI
Figure List VIII
Abbreviation IX
Introduction 1
Autophagy 1
Tumorigenesis 1
Autophagy and Tumorigenesis 2
Autophagy-independent Functions of Autophagy-related Genes (Atgs) 3
Specific Aim 4
Materials and Methods 6
I. Cell Lines and Cell Culture 6
II. Transient Transfection and Plasmids 6
III. Calcium Phosphate Transfection 6
IV. Western Blotting and Antibodies 7
V. MTT Assay 8
VI. Cell Viability Analysis 8
VII. BrdU Incorporation Assay 8
VIII. Colony Formation 9
IX. Cell Migration Assay 9
X. RNA Extraction 9
XI. RT-PCR 10
XII. Xenografted Tumor Mice Model 10
XIII. Statistical Analysis 11
Results 12
I. Mouse embryonic fibroblasts with various autophagy deficiency showed decreases cell growth and cell migration 12
II. Establishment of stable cell lines expressing Atg5 using Atg7-/- MEF autophagy-deficiency cell line 13
III. Over expression of Atg5 protein in Atg7-/- MEF cells increases cell growth and cell migration 14
IV. Atg5 increases anchorage-independent growth 15
V. Atg5 protein transiently induces tumor formation in xenograft NOD/SCID mice model 16
VI. Atg5 overexpression decreases cell proliferation and migration when autophagy was recovered 16
VII. -catenin is decreased and p-JNK is increased by Atg5 overexpression in Atg7-/- MEF cells 17
Discussion 19
References 25
Figures 30
Appendix 47

參考文獻 1 Mizushima N (2007). Autophagy: process and function. Genes Dev 21: 2861-2873.

2 Mizushima N, Yoshimori T, Ohsumi Y (2011). The role of Atg proteins in autophagosome formation. Annu Rev Cell Dev Biol 27: 107-132.

3 Russell RC, Yuan HX, Guan KL (2014). Autophagy regulation by nutrient signaling. Cell Res 24: 42-57.

4 Gillies RJ, Verduzco D, Gatenby RA (2012). Evolutionary dynamics of carcinogenesis and why targeted therapy does not work. Nat Rev Cancer 12: 487-493.

5 Hanahan D, Weinberg RA (2000). The hallmarks of cancer. Cell 100: 57-70.

6 Bergers G, Benjamin LE (2003). Tumorigenesis and the angiogenic switch. Nat Rev Cancer 3: 401-410.

7 Roy S, Debnath J (2010). Autophagy and tumorigenesis. Semin Immunopathol 32: 383-396.

8 Mizushima N, Levine B, Cuervo AM, Klionsky DJ (2008). Autophagy fights disease through cellular self-digestion. Nature 451: 1069-1075.

9 Liang XH, Jackson S, Seaman M, Brown K, Kempkes B, Hibshoosh H et al (1999). Induction of autophagy and inhibition of tumorigenesis by beclin 1. Nature 402: 672-676.

10 Kang MR, Kim MS, Oh JE, Kim YR, Song SY, Kim SS et al (2009). Frameshift mutations of autophagy-related genes ATG2B, ATG5, ATG9B and ATG12 in gastric and colorectal cancers with microsatellite instability. J Pathol 217: 702-706.

11 Ying SH, Liu J, Chu XL, Xie XQ, Feng MG (2016). The autophagy-related genes BbATG1 and BbATG8 have different functions in differentiation, stress resistance and virulence of mycopathogen Beauveria bassiana. Sci Rep 6: 26376.

12 Reggiori F, Monastyrska I, Verheije MH, Cali T, Ulasli M, Bianchi S et al (2010). Coronaviruses Hijack the LC3-I-positive EDEMosomes, ER-derived vesicles exporting short-lived ERAD regulators, for replication. Cell Host Microbe 7: 500-508.

13 Ishibashi K, Uemura T, Waguri S, Fukuda M (2012). Atg16L1, an essential factor for canonical autophagy, participates in hormone secretion from PC12 cells independently of autophagic activity. Mol Biol Cell 23: 3193-3202.

14 Lee IH, Kawai Y, Fergusson MM, Rovira, II, Bishop AJ, Motoyama N et al (2012). Atg7 modulates p53 activity to regulate cell cycle and survival during metabolic stress. Science 336: 225-228.

15 Zhao Z, Fux B, Goodwin M, Dunay IR, Strong D, Miller BC et al (2008). Autophagosome-independent essential function for the autophagy protein Atg5 in cellular immunity to intracellular pathogens. Cell Host Microbe 4: 458-469.

16 Maskey D, Yousefi S, Schmid I, Zlobec I, Perren A, Friis R et al (2013). ATG5 is induced by DNA-damaging agents and promotes mitotic catastrophe independent of autophagy. Nat Commun 4: 2130.

17 Saitoh T, Fujita N, Hayashi T, Takahara K, Satoh T, Lee H et al (2009). Atg9a controls dsDNA-driven dynamic translocation of STING and the innate immune response. Proceedings of the National Academy of Sciences of the United States of America 106: 20842-20846.

18 Wu SY, Lan SH, Cheng DE, Chen WK, Shen CH, Lee YR et al (2011). Ras-Related Tumorigenesis Is Suppressed by BNIP3-Mediated Autophagy through Inhibition of Cell Proliferation. Neoplasia 13: 1171-U1113.

19 Mori S, Chang JT, Andrechek ER, Matsumura N, Baba T, Yao G et al (2009). Anchorage-independent cell growth signature identifies tumors with metastatic potential. Oncogene 28: 2796-2805.

20 Ozeki N, Hase N, Hiyama T, Yamaguchi H, Kawai R, Kondo A et al (2015). Interleukin-1beta-induced autophagy-related gene 5 regulates proliferation of embryonic stem cell-derived odontoblastic cells. PLoS One 10: e0124542.

21 Wang C, Zhao Y, Su Y, Li R, Lin Y, Zhou X et al (2013). C-Jun N-terminal kinase (JNK) mediates Wnt5a-induced cell motility dependent or independent of RhoA pathway in human dental papilla cells. PLoS One 8: e69440.

22 Mikels AJ, Nusse R (2006). Purified Wnt5a protein activates or inhibits beta-catenin-TCF signaling depending on receptor context. PLoS Biol 4: e115.

23 Knudson AG (2001). Two genetic hits (more or less) to cancer. Nat Rev Cancer 1: 157-162.

24 Fearon ER, Vogelstein B (1990). A genetic model for colorectal tumorigenesis. Cell 61: 759-767.

25 Kuma A, Hatano M, Matsui M, Yamamoto A, Nakaya H, Yoshimori T et al (2004). The role of autophagy during the early neonatal starvation period. Nature 432: 1032-1036.

26 Takamura A, Komatsu M, Hara T, Sakamoto A, Kishi C, Waguri S et al (2011). Autophagy-deficient mice develop multiple liver tumors. Genes Dev 25: 795-800.

27 Wang L, Yao L, Zheng YZ, Xu Q, Liu XP, Hu X et al (2015). Expression of autophagy-related proteins ATG5 and FIP200 predicts favorable disease-free survival in patients with breast cancer. Biochem Biophys Res Commun 458: 816-822.

28 Rao S, Tortola L, Perlot T, Wirnsberger G, Novatchkova M, Nitsch R et al (2014). A dual role for autophagy in a murine model of lung cancer. Nat Commun 5: 3056.

29 Cho DH, Jo YK, Kim SC, Park IJ, Kim JC (2012). Down-regulated expression of ATG5 in colorectal cancer. Anticancer Res 32: 4091-4096.

30 Liu H, He Z, von Rutte T, Yousefi S, Hunger RE, Simon HU (2013). Down-regulation of autophagy-related protein 5 (ATG5) contributes to the pathogenesis of early-stage cutaneous melanoma. Sci Transl Med 5: 202ra123.

31 Li X, Li C, Zhu LH (2015). [Correlation of autophagy-associated gene Atg5 with tumorigenesis of prostate cancer]. Natl J Andro 21: 31-34.

32 Sun K, Deng W, Zhang S, Cai N, Jiao S, Song J et al (2013). Paradoxical roles of autophagy in different stages of tumorigenesis: protector for normal or cancer cells. Cell Biosci 3: 35.

33 Zhu N, Qin L, Luo Z, Guo Q, Yang L, Liao D (2014). Challenging role of Wnt5a and its signaling pathway in cancer metastasis (Review). Exp Ther Med 8: 3-8.

34 Valenta T, Hausmann G, Basler K (2012). The many faces and functions of beta-catenin. EMBO J 31: 2714-2736.

35 Botrugno OA, Fayard E, Annicotte JS, Haby C, Brennan T, Wendling O et al (2004). Synergy between LRH-1 and beta-catenin induces G1 cyclin-mediated cell proliferation. Mol Cell 15: 499-509.

36 Wagner RT, Xu X, Yi F, Merrill BJ, Cooney AJ (2010). Canonical Wnt/beta-catenin regulation of liver receptor homolog-1 mediates pluripotency gene expression. Stem Cells 28: 1794-1804.

37 Shtutman M, Zhurinsky J, Simcha I, Albanese C, D'Amico M, Pestell R et al (1999). The cyclin D1 gene is a target of the -catenin/LEF-1 pathway. Proceedings of the National Academy of Sciences 96: 5522-5527.

38 Zhang W, Chen X, Kato Y, Evans PM, Yuan S, Yang J et al (2006). Novel cross talk of Kruppel-like factor 4 and beta-catenin regulates normal intestinal homeostasis and tumor repression. Mol Cell Biol 26: 2055-2064.

39 Xing Y, Ma X, Guo H, Deng F, Yang J, Li Y (2016). Wnt5a Suppresses beta-catenin Signaling during Hair Follicle Regeneration. International journal of medical sciences 13: 603-610.

40 Rognoni E, Gomez C, Pisco AO, Rawlins EL, Simons BD, Watt FM et al (2016). Inhibition of beta-catenin signalling in dermal fibroblasts enhances hair follicle regeneration during wound healing. Development 143: 2522-2535.

41 Posthaus H (2002). beta-Catenin is not required for proliferation and differentiation of epidermal mouse keratinocytes. J Cell Sci 115: 4587-4595.
論文全文使用權限
  • 同意授權校內瀏覽/列印電子全文服務,於2021-07-20起公開。


  • 如您有疑問,請聯絡圖書館
    聯絡電話:(06)2757575#65773
    聯絡E-mail:etds@email.ncku.edu.tw